Human rhinoviruses (HRVs) are major causative agents of the common cold, one of the most frequent human infections. They are also the most common pathogens associated with asthma exacerbations. Despite the medical importance of rhinoviruses, the pathogenesis of rhinovirus infection is poorly understood because a convenient animal model is not available. The experiments in this proposal address a wide range of experimental questions on the biology of rhinovirus, encompassing replication in cell culture, the molecular basis of host range, innate responses to infection, and establishing replication in mice. These goals will be accomplished through the following four specific aims. 1. Determine the role in rhinovirus replication of cell proteins that interact with the viral 2B and 3A proteins. We have shown that the 3A protein of HRV39 interacts with GCP60 and FinGERS, proteins involved in vesicle traffic, and with an enzyme involved in lipid biosynthesis, CDIPT (phosphatidylinositol synthase). Cell proteins that interact with 2B will also be identified. We will assess the role of these cell proteins in viral replication, alteration of membrane ultrastructure, and inhibition of ER-to-Golgi traffic by reducing the levels of these proteins in the cell, interfering with their function with dominant negative forms of the proteins, or by producing non-interacting altered viruses. 2. Determine the role of the rhinovirus IRES in viral host range. We have found that the IRES of HRV2 does not mediate translation of a reporter protein in most organs of adult mice, including the lung. However, HRV2 IRES-mediated translation takes place in many organs of neonatal mice. Experiments in this specific aim are designed to determine whether the IRES of RV16, RV39, and RV1A can function in cells of the mouse lung. These cell cultures will be used to determine if specific structured regions of the IRES prevent internal initiation in primary lung cells from adult mice. Experiments are also planned to determine whether the lack of HRV2 IRES-mediated activity in cultured mouse cells is due to an inhibitor or absence of a required protein, and experiments to identify such factors are proposed. 3. Determine the role of pattern recognition molecules in rhinovirus replication. Experiments in this specific aim aredesigned to determine the role of specific pattern recognition molecules in rhinovirus replication. We will determine the roles of RIG-I, MDA-5, TLR3, and TLR7 in sensing rhinovirus replication in cultured cells. We have found that RIG-I and MDA-5 are cleaved in cells infected with rhinoviruses. Experiments are planned to determine whether cleavage of these sensor molecules benefits viral replication. In addition, we will determine whether rhinoviruses can replicate in dendritic cells, and whether TLR3 or TLR7 plays a role in sensing the viral genome in these cells. The identification of cell proteins that play a role in rhinovirus replication may reveal new targets for therapeutic intervention of the common cold.